SUPPLEMENTARY MATERIAL “An in vivo biosensor for neurotransmitter release and in situ receptor activity”

FIGURE CAPTIONS

Supplementary Figure 1. M1-CNiFER Dynamics in calcium-free media. These data

demonstrate that the tonic M1-CNiFER response is dependent upon calcium in the

media. (a) M1-CNiFERs respond with typical phasic and tonic components to 30 nM

acetylcholine in the constant presence of calcium. (b) M1-CNiFERs respond with only

the phasic component in calcium free media. Calcium returned to the media in the

continued presence of 30 nM acetylcholine results in a response with phasic and tonic

components, though possibly a slower phasic onset. (c) Calcium withdrawal from media

during the M1-CNiFER tonic response abolishes the response.

Supplementary Figure 2. Repetitive stimulation of M1-CNiFERs with pulses of acetylcholine. Two second pulses of 300 nM acetylcholine pulses, with a 30 second

interstimulus interval, were applied. Stabilization occurs at to an asymptotic value of 0.3-

times the initial change with a time-constant or 70 s.

Supplementary Figure 3. M1-CNiFERs respond to slowly-increasing concentrations of acetylcholine. A linear gradient of acetylcholine from 0 to 10 nM

was presented to M1-CNiFERs on a period of 1200 s; the gradient was calibrated by

concurrent measurements of co-dissolved alexa-594. The M1-CNiFER response (black

trace) to the slowly-increasing acetylcholine lacks a prominent phasic component as is

characteristic of the response to a step of acetylcholine (Fig. 1b). The M1-CNiFER

response to a bolus of 10 nM acetylcholine applied at the end of the experiment is

largely unchanged from that applied prior to start of the gradient. Note that there is a

small offset at the end of the experiment of possible technical origin; a similar level of

drift is seen without the addition of acetylcholine (gray trace).

Supplementary Figure 4. Atropine, clozapine and olanzapine suppress receptor- mediated responses in vitro. In Flexstation™ 3 assay, the M1-CNiFER FRET

response to 100 nM acetylcholine chloride is abolished by 100 nM atropine sulfate,

3 µM clozapine, and 3 µM olanzapine. All three effects are significant by t-test,

p < 0.001, n = 6 for each group. Bars represent standard error.

Supplementary Figure 5. M1-CNiFER response to acetylcholine is maintained

after brain implantation. M1-CNiFERs respond to intracortical 27 nl puffs of 1 mM acetylcholine chloride (cyan) but not PBS (grey). Time trace shows average response and standard error for 3 injections, same animal. Inset shows fractional change of 1/3

the area under the ∆R/R curve for 30 s after the stimulus as compared to that for 10 s

before the stimulus, for either acetylcholine or PBS (3 animals, 3 trials each). Bars

represent standard error.

Supplementary Figure 6. Intact vasculature surrounding M1- and control-CNiFER sites in the frontal cortex of a live rat implanted two days earlier. Image is a z-

projection spanning 140 µm from the cortical surface. Cerebrovasculature is visualized

by 500 µl intravenous injection of 5 % (w/w) fluorescein dextran (yellow). Visibility of the

fluorophore shows the vasculature is intact. M1-CNiFERs labeled in cyan (1,3), control-

CNiFERs labeled in red (2).

Supplementary Figure 7. Immunostain against GFAP reveals no astrocytic scar

around chronically implanted M1- and control-CNiFERs. In top image, mCherry and

TN-XXL fluorescence are overlayed on a brightfield 3,3'-diaminobenzidine α-GFAP stain

for astrocytes. Scale bar represents 200 µm. In bottom image, only TN-XXL

fluorescence is overlayed to better visualize the morphology of CNiFER cells. Scale bar

represents 50 µm. α-GFAP stain reveals cell bodies and long complex processes

apparent at the surface of the brain and in deeper layers at ~ 1 mm (see white arrows).

There is no astrocytic concentration (glial scar) along the implantation site. Functional

data with in vivo two photon microscopy is typically acquired at depths of 50 to 200 µm

below the pia, where few astrocytes in this section are present. Preparation was 2 d in

vivo; M1-CNiFER column extends to the cortical surface in a neighboring section (data

not shown).

Supplementary Figure 8. Clozapine suppresses NBM-evoked activation of ECoG, consistent with suppression of M1-CNiFER response. (a) Spectral analysis of

electrocorticogram reveals the hallmark of NBM-evoked cortical activation: a

suppression of power in the δ-band. Intraperitoneal clozapine (5 mg/kg) abolishes this

effect, consistent with its antimuscarinic properties and consistent with its effect on M1-

CNiFERs. Pre-NBM spectra in blue, post-NBM spectra in green. (b) Summary graph

showing statistical significance of NBM-evoked cortical activation as measured by

minus one times the logarithm of the inverse power of delta band,

-log[power in ECoG δ-band], and its modulation by clozapine. As expected, NBM

stimulation significantly decreases power in the δ-band (p = 0.005, t-test). In the

presence of clozapine, NBM stimulation mildly increases the power in the δ−band

(p = 0.04, t-test). Furthermore, clozapine slows the baseline spontaneous

electrocorticogram (p = 0.008, t-test). Bars represent standard error.

Supplementary Figure 9. Olanzapine suppression of NBM-evoked M1-CNiFER

response is not activity dependent. Intraperitoneal olanzapine (3 mg/kg) suppresses

the M1-CNiFER response to a single NBM stimulation delivered 20 minutes after

injection of the antipsychotic. Black vertical dashed lines represent 600 µA NBM

stimulations.

0 200 400 600 800 1000 1200

0 200 400 600 800 1000 1200

30 nM AChCa2+Ca2+ Ca2+ free

30 nM AChCa2+ Ca2+ free

0 200 400 600 800 1000 12000

1.0

0

1.0

0

1.0

Time (s)

Time (s)

Time (s)

(a)

(b)

(c)

30 nM AChCa2+

Supplemental figure 1. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

CN

iFE

R F

RE

Tra

tio, Δ

R/R

CN

iFE

R F

RE

Tra

tio, Δ

R/R

CN

iFE

R F

RE

Tra

tio, Δ

R/R

0 50 100 150 200 250 300 350 400

0

20

40

60

80

100

Time (seconds)

M1-

CN

iFE

R Δ

R/R

(%)

ACh (pulses, 30s isi)

Supplemental figure 2. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

[Ace

tylc

holin

e](µ

M)

M1-

CN

iFE

R F

RE

T S

igna

l, Δ

R/R

1.0

1.2

1.4

1.6

0

10

5

Time after initial bolus (s)

0 500 1000 1500 2000 2500 3000

Supplemental figure 3. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

100 nMACh

100 nMACh

+100 nMatropine

100 nMACh

+3 μM

olanzapine

100 nMACh

+3 μM

clozapine

p < 0.001

M1-

CN

iFE

R F

RE

T ra

tio, Δ

R/R

Supplemental figure 4. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

20 s

AChVehicle

CN

iFE

R F

RE

T ra

tio, Δ

R/R

1 2 31.01.11.21.31.4

ΔR

/R

puff

1.25

Time

Sample

Supplemental figure 5. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

200 μm

1 2 3

Supplemental figure 6 Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

control-CNiFERs

M1-CNiFERs

200 μm

50 μm

control-CNiFERs

Supplemental figure 7. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

0 1 2 3 4 5 6

post-NBM (n=10)pre-NBM (n=10)

post-NBM, after clozapine (n=8)pre-NBM, after clozapine (n=8)

Frequency [Hz]

Spe

ctra

l pow

er

−1.0

−0.5

0

0.5

1.0

1.5

Pre-NBMn = 30

Post-NBMn = 30

Pre-NBMn = 30

Post-NBMn = 30

Clozapine

p = 0.04

p = 0.008

p = 0.005

a b

Z-sc

ore

of -l

og[E

CoG

δ-b

and]

Supplemental figure 8. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld

2 min

Vehicle

NBM stimulus600 μA

Olanzapine3 mg/kg

NBM stimulus600 μA

10 s10 s

NBM stimulus600 μA

NBM stimulus600 μA

0.05

CN

iFE

R F

RE

T ra

tio, Δ

R/R

Time

Supplemental figure 9. Q.-T. Nguyen*, L F. Schroeder*, M. Mank, A. Muller, P. W. Taylor, O. Griesbeck and D. Kleinfeld